Research in the Zakrzewski Lab
Harnessing the Thymus for Targeted Cancer Immunotherapy
B6 mice were irradiated with 850 cGy and transplanted with 100,000 B6.CD45.1 Lin- BM cells. Mice received 3,000 B6.LUC.Thy1.1 LSK cells via ultrasound-guided intrathymic injection 2 hours after irradiation. Control mice received no injections. In vivo BLI was performed at the indicated time points after irradiation. (n=4)
Tuckett AZ et al. Blood 2012; 123: 2797-2805
Although the thymus is an obvious target organ for immunotherapeutic strategies, thymus-based approaches have traditionally been underutilized in clinical trials to improve immunity. As a way to address this need, we developed a minimally invasive, accurate, and rapid method for intrathymic injection based on image-guided free-hand injection. Due to the unique capacity of the thymic microenvironment to impose a T-cell lineage fate on multipotent hematopoietic stem and progenitor cells, intrathymic injection of bone marrow or cord blood-derived HSPCs holds significant promise for immunotherapy and clinical translation. Importantly, this approach has the benefit of being able to generate long-lasting immunity to specific antigens (pathogenic or oncogenic) through gene manipulation.
We recently demonstrated that a single intrathymic injection of HSPCs harboring a transgenic T-cell receptor promotes long-lasting antigen-specific T-cell immunity. We are currently developing a method for in vivo generation of T cells with desired specificities and functions by subjecting multipotent HSPCs to two interventions: genetic engineering (to introduce antigen specificity), and image-guided injection into the thymus. Since CAR-transduced HSPCs developing into T cells may run into road blocks during their journey through the thymus (specifically with regard to negative selection), we are employing an arsenal of alternate strategies to overcome the relevant biological check points. This project will facilitate the implementation of novel immunotherapeutic approaches designed to benefit patients in need of long-term control of high-risk malignancies.
Immunobioengineering of Tissue Constructs Supporting T-Cell Development
Laser scanning confocal microscopy of thymic organoid on day 10 of in vitro culture. Thymic epithelial cells were analyzed for cytokeratin (CK) 8 and CK 14.
We are developing implantable biomimetic tissue constructs designed to improve T-cell function and optimize antitumor immunity. Our approaches utilize thymic epithelial cells and other cell sources, biodegradable polymer scaffolds, and hydrogel systems to produce artificial microenvironments that serve as implantable hematopoietic precursor cell niches. A major focus of this research program is to develop methods to supplement bioengineered tissue constructs with various growth factors, cytokines, and chemokines required for T-cell development. In addition to in vitro testing, we routinely utilize preclinical mouse models for in vivo efficacy testing of our artificial thymus platforms.
Targeting NF-κB and Oxidative Stress in Multiple Myeloma
(LEFT) IT compound X inhibits expression of the Nf-kB target gene interleukin 10 (IL-10). TMD8, HBL1, and U266 cells were incubated for 24 hours in the presence of empty vehicle or IT compound X (4 and 6 µm). IL-10 concentrations in the culture media were analyzed by ELISA. Mean and SEM of relative IL-10 levels (percentage of vehicle) are presented.
(RIGHT) IT-901 increases ROS levels in lymphoma cells but not in normal lymphocytes. Cells were incubated for 24 hours on the presence of empty vehicle or 4µm of IT-901. Total ROS levels were quantified for DCFDA assay. Fold changes over baseline (vehicle treatment is presented).
Oxidative stress and NF-κB play differential roles in healthy and diseased cells. Manipulation of the cellular redox balance has the potential to be therapeutically exploitable in a wide range of cancers, including lymphomas and myelomas. Constitutive activity of NF-κB family members such as c-Rel has been associated with oncogenesis, and NF-κB is also an important part of the oxidative stress response.
We previously described a novel small molecule compound combining inhibition of NF-κB DNA binding with the capacity to modulate the redox balance of B-cell lymphoma cells, resulting in potent immunomodulatory properties and anti-lymphoma activity. The overarching goal of this research program is to build on this work and identify through structure activity relationship (SAR) testing a small molecule with optimized physicochemical properties, pharmacokinetics (PK), potency, and safety profile. We are performing mechanistic and drug development studies in parallel with the goal to improve the understanding of the impact of NF-κB inhibition and oxidative stress modulation on lymphoid malignancies while expediting the development of a small molecule inhibitor drug, in collaboration with ImmuneTarget Inc.